Integrated molecularly imprinted polymer and surface-enhanced raman (SERS) substrates: method and applications

Abstract

Provided herein is technology relating to a method for selectively detecting a target analyte, such as environmental pollutants in a sample; a method of detecting surface-enhanced Raman spectroscopy (SERS) signals of a molecularly imprinted polymers (MIPs) matrix binding to a target analyte; a composition comprising a molecularly imprinted polymers (MIPs) matrix, wherein the MIP matrix comprises noble metal nanoparticles or hybrid plasmonic nanoparticles and an activated recognition cavity specific to a target analyte; and a method for in-situ manufacturing a molecularly imprinted polymers (MIPs) matrix comprising noble metal nanoparticles or hybrid plasmonic nanoparticles.

Description

[0001] Docket No.: UNCG-532-PCT

[0002] INTEGRATED MOLECULARLY IMPRINTED POLYMER AND SURFACE-ENHANCED RAMAN (SERS) SUBSTRATES: METHOD AND APPLICATIONS

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 735,878 filed on December 18, 2024.

[0005] FIELD

[0006] Provided herein is technology relating to a method for selectively detecting a target analyte, such as environmental pollutants in a sample; a method of detecting surface-enhanced Raman spectroscopy (SERS) signals of a molecularly imprinted polymers (MIPs) matrix binding to a target analyte; a composition comprising a molecularly imprinted polymers (MIPs) matrix, wherein the MIP matrix comprises noble metal nanoparticles or hybrid plasmonic nanoparlicles and an activated recognition cavity specific to a target analyte; and a method for in-situ manufacturing a molecularly imprinted polymers (MIPs) matrix comprising noble metal nanoparticles or hybrid plasmonic nanoparticles.

[0007] BACKGROUND

[0008] Environmental pollution, with its negative implications, continues to affect sustainability, pose public health threats and challenges for many global economies. To accurately determine the levels of environmental pollutants, such as pesticide residues in the environment, it is essential to develop rapid and precise sensing techniques, such as chemosensors, to safeguard the ecosystem and for public health safety. Conventional analytical methods such as high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) have a high cost, time-consuming nature, and technical complexity [1], making them less attractive for rapid sensing and in-field monitoring applications [2, 3]. Thus, there is a need for improved sensing technology for detecting and / or monitoring environmental pollutants in complex matrices, such as drinking water and tap water.

[0009] SUMMARY

[0010] The subject matter disclosed herein provides a method for selectively detecting a target analyte in a sample, the method comprising: providing a composition comprising a molecularly imprinted polymers (MIP) matrix, wherein the MIP matrix comprises a) noble metal nanoparticles or hybrid plasmonic nanoparticles and b) activated recognition cavities comprising binding sites and cavities complementary to a target analyte; contacting the composition with the target analyte; and Docket No.: UNCG-532-PCT detecting binding of the MIP matrix to the target analyte in a sample, thereby detecting the target analyte in the sample. Advantageously, in some aspects, the MIP matrix comprises uniformly dispersed noble metal nanoparticles or uniformly dispersed hybrid plasmonic nanoparticles. In some aspects, the noble metal nanoparticles or the hybrid plasmonic nanoparticles are encapsulated on the surface of the MIPs matrix and embedded within the MIPs matrix. In some aspects, the noble metal nanoparticles are silver nanoparticles or gold nanoparticles. In some aspects, the hybrid plasmonic nanoparticles comprise noble metal nanoparticles and carbon nanoparticles.

[0011] In some aspects, the composition is a surface -enhanced Raman scattering (SERS) substrate and wherein detecting binding of the MIP matrix to the target analyte in a sample comprises detecting Raman signals. In some aspects, the Raman signals are surface-enhanced Raman spectroscopy (SERS) signals. In some aspects, the SERS signals comprise a fingerprint of the binding of the MIP matrix to the target analyte. In some aspects, the fingerprint comprises an amplified SERS signal of the MIP matrix and / or an amplified SERS signal of the target analyte. In some aspects, the intensity of amplified SERS signal of the MIP matrix and / or the intensity of the amplified SERS signal of the target analyte is directly proportional to the concentration of the target analyte in the sample.

[0012] In some aspects, the target analyte is an environmental pollutant. In some aspects, the target analyte is an organophosphate pesticide, herbicide, a PFAS molecule, or a heavy metal ion. In some aspects, the target analyte is malathion, chlorpyrifos, dimethoate, or parathion. In some aspects, the target analyte is present in the sample at a low concentration ranging from 0.005 pg / nil to 50 JJ g / inl . In some aspects, the target analyte is present in the sample in a concentration ranging from 0.005 pg / ml to 40 pg / ml, 0.005 pg / ml to 30 pg / ml, 0.005 g / ml to 20 pg / ml, or 0.005 pg / ml to 10 pg / ml. In some aspects, the minimum detection limit is at least 0.005 pg / ml. In some aspects, the method further comprises recovering the target analyte with a recovery rate greater than 90%, 91%, 92%, or 93%. In some aspects, the sample comprises complex environmental matrices. In some aspects, the sample is tap water, drinking water, river water, urine, agroecosystem sediments, a fruit, or a vegetable.

[0013] The subject matter disclosed herein also provides a composition comprising a molecularly imprinted polymers (MIPs) matrix, wherein the MIP matrix comprises: a) noble metal nanoparticles or hybrid plasmonic nanoparticles, and b) an activated recognition cavity comprising binding sites and a structural shape complementary to a target analyte. Advantageously, in some aspects, the MIP matrix comprises uniformly dispersed noble metal nanoparticles or uniformly dispersed hybrid plasmonic nanoparticles. In some aspects, the noble metal nanoparticles or the hybrid plasmonic nanoparticlcs arc encapsulated on the surface of the MIPs matrix and embedded within the MIPs matrix. In some aspects, the noble metal nanoparticles are silver nanoparticles or gold nanoparticles. Docket No.: UNCG-532-PCT

[0014] In some aspects, hybrid plasmonic nanoparticles comprise noble metal nanoparticles and carbon nanoparticles.

[0015] In some aspects, the noble metal nanoparticles are silver nanoparticles, and the MIP matrix comprises activated recognition cavities complementary to malathion. In some aspects, the template target analyte is an organophosphate pesticide, herbicide, a heavy metal ion, or a PF AS molecule. In some aspects, the template target analyte is an environmental pollutant.

[0016] The subject matter disclosed herein also provides a method for in-situ manufacturing a molecularly imprinted polymers (MIPs) matrix comprising noble metal nanoparticles, the method comprising: providing a homogenous mixture of a noble metal precursor molecule, a template target analyte, a ftmctional monomer, a crosslinker, and an initiator; polymerizing / solidifying the homogenous mixture to produce a molecularly imprinted polymers (MIP) matrix comprising the noble metal precursor molecule and the template target analyte; reducing the noble metal precursor molecule into a noble metal nanoparticle in-situ within the MIP matrix, wherein the MIP matrix comprises noble metal nanoparticles or hybrid plasmonic nanoparticles; removing the template target analyte from the MIP matrix, wherein the MIP matrix comprises activated recognition cavities specific to the target analyte. Advantageously, in some aspects, the MIP matrix comprises uniformly dispersed noble metal nanoparticles or uniformly dispersed hybrid plasmonic nanoparticles. In some aspects, the noble metal nanoparticles or the hybrid plasmonic nanoparticles are encapsulated on the surface of the MIPs matrix and embedded within the MIPs matrix.

[0017] In some aspects, the functional monomer is methyl methacrylate (MMA), acrylamide, acrylic acid, glycidyl methacrylate (GMA), fumaric acid, itaconic acid or vinyl siloxanes. In some aspects, the cross-linker is ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TRIM), divinylbenzene (DVB), polyethylene glycol dimethacrylate (PEGDMA), tetraethylene glycol dimethacrylate (TEGDMA), or ethylene glycol diacrylate (EGDA). In some aspects, the initiator is 2,2'-azobisisobutyronitrile (AIBN), benzoyl pereoxide (BPO), azobisdimethylvaleronitrile (ABDV), or 2,2’-Azobisisovaleronitrile (AIVN). In some aspects, the functional monomer is methyl methacrylate (MMA), the initiator is 2,2'-azobisisobutyronitrile (AIBN), the cross-linker is ethylene glycol dimethacrylate (EGDMA), the noble metal precursor molecule is silver nitrate, and / or the noble metal nanoparticle is a silver nanoparticle is silver nitrate.

[0018] In some aspects, the homogenous mixture further comprises a reducing agent. In some aspects, the reducing agent is NaBI I4. In some aspects, the template target analyte is an organophosphate pesticide, herbicide, a heavy metal ion, or a PFAS molecule. In some aspects, the template target analyte is an environmental pollutant. In some aspects, removing the template target analyte from the MIP matrix comprises reacting the MIP matrix with an organic solvent. Docket No.: UNCG-532-PCT

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] These and other features, aspects, and advantages of the present technology will become better understood with regal'd to the following drawings:

[0021] FIG. 1 is a schematic illustration of the AgNPs @ MIP synthesis for a SERS sensor substrate. The size ratio of components is not in accordance with a real case. The plasmonic nanoparticles are not limited to AgNPs. They can be gold (AuNPs), AgNPs, or hybrid plasmonic nanoparticles comprising nano-carbonaceous materials.

[0022] FIG. 2 shows the chemical structure of the MIP agents employed for using malathion as the template molecules in AgNPs @ MIP synthesis.

[0023] FIG. 3 is a TEM image of the in-situ synthesized spherically shaped AgNPs (~20 nm) produced in the AgNPs @ MIP.

[0024] FIG. 4 shows scanning electron microscopy (SEM) images of the non-eluted AgNPs@MlP

[0025] (A), eluted AgNPs@MIP (B), non-eluted AgNPs@NIP (C), and eluted AgNPs@NIP (D) respectively, where NIP represents the same polymer synthesized without the template molecules. The effect of organic solvent in the template removal process creates cavities in the MIP thus enhancing its binding affinity for the target analyte malathion.

[0026] FIG. 5 illustrates the UV-Vis analysis before and after the template removal process and the formation of AgNPs within the MIP or NIP platform. (A): Shows the UV-vis peaks of the pure malathion and the AgNPs@MIP, before and after the reduction of AgNP in the presence of NaBH4.

[0027] (B): UV-Vis of the AgNPs <4 MIP and AgNPs@NIP confirming the doping of AgNPs into the MIP or NIP platform after the successful removal of the malathion template. (C and D): The UV-vis of the AgNPs@MIP or AgNPs@NIP during the first and second stage elution process of the MIP or NIP platform with organic solvents (methanol and acetic acid).

[0028] FIG. 6 demonstrates kinetic adsorption of the AgNPs @MIPs and the AgNPs @ NIP with variable concentrations of malathion at a given time.

[0029] FIG. 7 demonstrates X-ray diffraction (XRD) of the AgNPs@MIP shows the crystalline diffraction peaks of AgNPs doped into the fabricated AgNPs @ MIP. Blue lines represent the diffraction peaks of the AgNPs and the red line represents XRD spectrum of the AgNPs @ MIP.

[0030] FIG. 8 shows the FTIR spectra of the AgNPs@MIPs, AgNPs@NIPs and other chemical agents employed for the synthesis showing the characteristics of functional groups that were identified.

[0031] FIG. 9 shows the FTIR spectra of the synthesized malathion AgNPs@MIP and AgNPs@NIP.

[0032] FIG. 10 shows Raman spectra of pure malathion, malathion AgNPs @ MIP (AgNPs @ MIP after template removal), AgNPs @ NIP (non-imprinted polymer), AgNPs @ NIP (AgNPs @ NIP Docket No.: UNCG-532-PCT incubated with 10 pg / ml) and malathion AgNPs@MIP (AgNPs@MIP incubated with 10 pg / ml malathion).

[0033] FIG. 11 shows Raman spectru of pure malathion (a), SERS spectrum of eluted malathion AgNPs @ MIP (b), SERS spectra of the eluted AgNPs @ MIP after incubating with malathion solution at concentrations of 0.005 pg / ml (c), 0.05 pg / ml (d), 0.5 pg / ml (e), 5 jjg / rnl (f), and 50 pg / ml (g), respectively with SERS intensity of malathion detected at 1580 cm1by the AgNPs@MIP.

[0034] FIG. 12 shows the linear correlation of the logarithmic peak intensity at 1580 cm1versus the logarithmic concentrations of malathion

[0035] FIG. 13 shows the Raman spectra showing the selectivity of (A): SERS spectra of AgNPs @ MIP incubated with a blend of malathion and dimethoate (5 pg / ml) solution, AgNPs @ MIP incubated with malathion, and malathion solution only (no AgNPs@MIP), and (B) SERS spectra of AgNPs@MlP incubated mixed malathion and parathion, AgNPs@MlP incubated with malathion, and parathion only solution (no AgNPs @ MIP).

[0036] FIG. 14 shows the Raman spectra showing the (A): SERS spectra of AgNPs@MIP incubated with spiked drinking water at 20 pg / ml and 1 pg / ml of malathion solution respectively and (B): SERS spectra of AgNPs@MIP incubated with spiked tap water at 20 pg / ml and 1 pg / ml of malathion solution respectively.

[0037] FIG. 15 is a schematic illustration of the setup for AgNPs@MIP synthesis (in figure AgNPs @ MIP = AgNPs @ MIP.

[0038] FIG.16 shows the chemical structure of the MIP agents employed for using dimethoate as template in AgNPs @ MIP synthesis.

[0039] FIG. 17 shows the TEM image of the in-situ synthesized AgNPs produced in the AgNPs@MIP using dimethoate template.

[0040] FIG. 18 shows the SEM images of the dimethoate AgNPs@MIP (A), and (B) AgNPs@NIP (no template molecules).

[0041] FIG. 19 illustrates the UV-Vis analysis confirming the template removal process and the formation of AgNPs within the MIP platform. (A): the UV-vis peaks of pure dimethoate and the doping confirmation of AgNPs in the MIP, and (B): UV-Vis of the AgNPs@MIP after the elution process with the designated organic solvents to remove template.

[0042] FIG. 20 shows kinetic adsorption of the AgNPs @MIPs and the AgNPs @ NIP with variable concentrations of dimethoate at a given time.

[0043] FIG. 21 shows X-ray diffraction (XRD) of the dimethoate AgNPs@MIP that depicts significant diffraction peaks of AgNPs doped into the AgNPs @ MIP. The red line represents the dimethoate AgNPs @ MIP and the dark lines indicate the diffraction peaks of the AgNPs. Docket No.: UNCG-532-PCT

[0044] FIG. 22 shows the FTIR spectra of the AgNPs@MIPs (eluted), the pure dimethoate molecule and the functional monomer (methyl acrylic acid) and their characteristic functional groups.

[0045] FIG. 23 shows the FTIR spectra of the dimethoate AgNPs@MIP that show peaks after adsorption with 5 g / ml and 20 pg / ml of dimethoate solution.

[0046] FIG. 24 shows the FTIR peaks of the AgNPs@NIP after incubation with 5 pg / ml and 20 pg / ml of dimethoate solution.

[0047] FIG. 25 shows (A) Raman spectra of pure dimethoate and (B) silver nanoparticles (AgNPs)

[0048] FIG. 26 shows (A) Raman spectra of pure dimethoate, the AgNPs@MIP only, AgNPs@NIP treated with 10 pg / ml dimethoate, and the AgNPs@MIP treated with 10 pg / ml dimethoate, (B) SERS spectra of dimethoate AgNPs @MIP treated with dimethoate solution at varying concentrations of 0.005 pg / ml to 20 pg / ml, with the distinct SERS intensity band of dimethoate detected at 1587 cm1by the AgNPs @MIP, (C) the linear calibration between the SERS intensity at 1587 cm'1and the concentration of dimethoate within the range 5 to 0.005 pg / ml.

[0049] FIG. 27 shows the selectivity of the dimethoate AgNPs@MIPthat shows the characteristic SERS intensity at 1587 cm-1of (A) dimethoate and chlorpyrifos and (B) dimethoate and fenthion all at 5 pg / ml concentration respectively.

[0050] FIG. 28 shows Raman spectra showing the (A), SERS spectra of dimethoate AgNPs @MIP incubated with spiked apple juice at 10 pg / ml and 0.5 pg / ml) of dimethoate solution respectively and (B) SERS spectrum of dimethoate AgNPs @MIP incubated with spiked orange juice at 10 pg / ml and 0.5 pg / ml) of dimethoate solution respectively. Also, the evaluated AgNPs @MIPs demonstrated excellent recoveries as depicted in Table 1.

[0051] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.

[0052] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on.” Docket No.: UNCG-532-PCT

[0053] DETAILED DESCRIPTION

[0054] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0055] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0056] “Molecularly imprinted polymers (MIPs) matrix” as used herein refers to a synthetic polymer platform as described herein.

[0057] “Activated recognition cavity” as used herein refers to a cavity of a M1P matrix comprising binding sites and a structural shape complementary to a target analyte. The acli vated recognition cavity is formed, for example, by methods described herein, e.g. from a structural imprinted template molecule that is subsequently removed. The activated recognition cavity is a receptor that specifically binds to a target analyte.

[0058] “Noble metal nanoparticles” and “hybrid plasmonic nanoparticles” as used herein refers to plasmonic nanoparticles. Examples of noble metal nanoparticles include silver nanoparticles, gold nanoparticles, or copper nanoparticles. Examples of hybrid plasmonic nanoparticles include nanoparticles comprises noble metal nanoparticles and carbon nanoparticles.

[0059] “Nanoparticles” (NPs) as used herein refers to particles having a spherical, cubic or other shape and has a size ranging from 1-1000 nm. The nanoparticles may have a spherical, rod, oval, octahedral, hexgonal, cubic, triangular, branched, or hollow shell shape. Examples include spherical nanoparticles that are about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm in diameter. Further examples include spherical nanoparticles that are about 10, 11 , 12,13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nm in diameter.

[0060] “Functional monomer” as used herein refers to one of the components in the MIP matrix synthesis forming the cavity for molecule recognition. Examples of functional monomers include methyl methacrylate (MM ), acrylamide, acrylic acid, glycidyl methacrylate (GM A), fumaric acid, itaconic acid, silanes, or vinyl siloxanes. In some aspects, the functional monomers have affinity for the target analyte.

[0061] “Cross-linker” as used herein refers to a chemical agent in the MIP matrix synthesis to covalently bond the monomers to form a polymer structure. Examples of cross-linkers include ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TRIM), Docket No.: UNCG-532-PCT divinylbenzene (DVB), polyethylene glycol dimethacrylate (PEGDMA), tetraethylene glycol dimethacrylate (TEGDMA), or ethylene glycol diacrylate (EGDA).

[0062] “Initiator” as used herein refers to a chemical agent that activates or initiates and promotes the MIP synthetic reaction. Examples of initiators include 2,2'-azobisisobutyronitrile (AIBN), benzoyl pereoxide (BPO), azobisdimethylvaleronitrile (ABDV), or 2,2’-Azobisisovaleronitrile (AIVN).

[0063] “Organic solvent” as used herein refers to solvents that contain carbon and oxygen.

[0064] Examples of organic solvents includes acetone, ethanol, acetonitrile (CAN), and hexane.

[0065] “Raman signal” or “Raman scattering signal” as used herein refers to a vibrational Raman spectrum that results from Raman scattering, a process that happens when light interacts with molecular vibration in a substance.

[0066] “Surface-enhanced Raman spectroscopy (SERS) signals” as used herein refers to the enhancement of a Raman scattering signal from molecules or a substance near to nanostructured metallic surfaces, usually made of noble metals, by the surface plasmon resonance (termed as nano- plasmonics).

[0067] “Fingerprint” as used herein refers to a substance’s unique Raman scattering signal that can differentiate the substance from other molecules. For example, a “fingerprint of the binding of the MIP matrix to the target analyte” comprises a Raman scattering signal which arises from the complex structure of the MIP matrix cavity as a receptor binding to the target analyte molecule.

[0068] “Environmental pollutant” as used herein refers to a substance introduced into the environment and causes adverse changes to health, environment, or ecosystem. Examples of environmental pollutants include water pollutants, chemical pollutants, and heavy metals, and microplastics. Examples of environmental pollutants includes pesticide residues, such as organophosphate pesticides, herbicides, carbamates, and heavy metal ions. Examples of organophosphate pesticides include malathion, chlorpyrifos, dimethoate, parathion diazinon, ethoprop, tribufos, phosmet, fonofos, parathion, diazinon, dichlorvos, fenitrothion, tetrachlorvinphos, azamethiphos, azinphos-methyl, phosalone, fenthion, dichlorofenthion, demton-methyl, and methyl parathion. Examples of heavy metal ions include mercury, lead, chromium, copper, manganese lead, arsenic, cadmium, copper, manganese, chromium, nickel, mercury, antimony, and selenium.

[0069] “Recovery rate” as used herein refers to a testing result / concentration of a spiked analyte as the percentage of the originally prepared concentration, which indicates the accuracy of the analysis using the sensor in detection. Docket No.: UNCG-532-PCT

[0070] “Complex environmental matrices” as used herein refers to environmental compositions comprising multiple analytes. Examples of complex environmental matrices include water, soil, dust, biota nature water or juices, that are made up of the original components without prelrealmenl.

[0071] Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

[0072] Molecularly imprinted polymers (MIPs) embedded with silver nanoparticles (AgNPs@MIPs) were synthesized for the selective detection of environmental pollutants (such as organophosphate pesticides, herbicides, heavy metal ions, PFAS) by the precipitation polymerization technique using methyl methacrylate (MMA) as a functional monomer, and target molecules (an OPP or metal ion) as a template molecule, ethylene glycol dimethacrylate (EGDMA) as cross-linker, 2,2'- azobisisobutyronitrile (AIBN) (AIBN) as an initiator, and the AgNPs as excellent surface-enhanced Raman scattering (SERS) substrates. The platform of AgNPs @ MIPs was well characterized regarding its morphology, structure, and spectroscopic features. Our results confirmed that the AgNPs @ MIPs specifically recognize the template molecules from complex matrices such as in drinking and tap water, juice and other water samples. The intensity of the SERS characteristic peak of the target analyte molecules caught in AgNPs @MIP was proportional to the evaluated concentrations, with a broad linear range, such as 0.005 to 5 pg / ml and a limit of detection (LOD) of 0.005 pg / inl and lower. The recovery rates and relative standard deviation for the detection of one representative OPP, e.g., malathion, spiked in the water samples were ranging from 93% to 100% and from 0.05% to 0.71%, respectively. Overall, the novel synergistic SERS-MIP method has demonstrated superior advantages and novelty in highly selective, sensitive, easy and quick binding and detection of OPPs and metal ions, promising for development of a sensing technology for monitoring many other organophosphate compounds and other pollutants for environmental monitoring and agriculture applications.

[0073] Molecular imprinting involves the polymerization of functionalized monomers in the presence of a template molecule. The resulting polymerized functional monomers present a complementary conformation to the template molecule and provide chemical interaction with its functional groups. A subsequent release of the template molecule leaves behind a polymeric activated recognition cavity with the desired shape and chemical functionality that is substantially complementary to the template molecule. The template molecule is selected based on the target molecule to be detected. In some aspects, the target analyte is used as a template molecule. Because the template molecule shares the same structure as the target molecule, the polymeric activated recognition cavity with the desired shape and chemical functionality that is substantially Docket No.: UNCG-532-PCT complementary to the template molecule will also be substantially complementary to the target molecule.

[0074] A template molecule is temporarily bound or coupled to the surface of a nanostructure. The template molecule used for molecular imprinting is selected based on the target molecule desired to be detected using the plasmonic nanotransducers. In some aspects, the target analyte is used as a template molecule. In one aspect, a cross-linker can be used to temporarily bind the template molecule to the surface of the nanostructure. The template molecule can then interact with the functional monomers that are polymerized on the nanostructure and surround the template molecule. Upon removal of the template molecule an activated recognition cavity is formed by the polymer with the desired shape and chemical functionality that is substantially complementary to the template molecule. Because the template molecule used to form the act i vated recognition cavity matches the target molecule desired to be detected, the activated recognition cavity is also substantially complementary to the target molecule.

[0075] EXAMPLES

[0076] Example 1: A Silver Molecularly Imprinted Polymer Surface-Enhanced Raman (SERS) Substrate: Synthesis, Characterization, and Applications for Malathion Detection LI Overview

[0077] Environmental pollution, with its negative implications, continues to affect sustainability, pose public health threats and challenges for many global economies [4,5], The role of human activities in the cycle of environmental pollution cannot be overemphasized, as pollutants are introduced into water bodies, the air and land [6], Malathion, one of the predominantly used organophosphate pesticides (OPPs) is a broad-spectrum agent widely used in agriculture to control pests and to ensure agricultural productivity [7]. However, despite the efficiency of pesticides, it has been established that less than 0.1 % of pesticides applied reach their intended targets, resulting in significant environmental pollution from pesticide residues [8], For example, malathion residues have been detected in several environmental matrices, including fruits and vegetables, drinking water, river water, agroecosystem sediments, and urine [7], Therefore, to accurately determine the levels of pesticide residues in the environment, the development of it is essential to develop rapid and precise sensing techniques, such as chemosensors, to safeguard the ecosystem and for public health safety [9,10].

[0078] The detection of pollutants such as pesticide residues represents a significant breakthrough that sensor technology can provide in addressing the ongoing global environmental pollution crisis [2], Although conventional analytical methods such as high-performance liquid chromatography Docket No.: UNCG-532-PCT

[0079] (HPLC), and gas chromatography-mass spectrometry (GC-MS) have been extensively employed for pesticide detection, their high cost, time-consuming nature, and technical complexity

[0001] make them less attractive for rapid sensing and in-field monitoring applications [2,3], Consequently, a class of chemosensors known as molecularly imprinted polymers (MIPs) have shown promising and attractiveness for detecting pesticide residues such as malathion detection in complex environmental matrices due to their specificity, sensitivity, and selectivity

[0010] .

[0080] MIPs are generally classified as polymeric materials that mimic the functionality of molecular recognition (e.g., antibody-antigen interactions). They are primarily fabricated through an imprinting technique that distinctively binds to templated molecules with a predefined specificity and selectivity for a target analyte [10-12], Consequently, the potential of MIP-based chemosensors for environmental applications and trace-level detection of pollutants in complex matrices, without the need of pre -treatment, paves the way for breakthroughs in rapid and real-time monitoring of in-situ contaminated samples

[0013] .

[0081] Additionally, the advent of surface-enhanced Raman spectroscopy (SERS) as a powerful, non-destructive sensing technique with enhanced sensitivity and relatively quick analysis time has attracted significant attention from many scientists [14,15], SERS substrates, such as AgNPs or noble metals, hold promising potential of actively transmitring Raman signals and detecting analytes at trace levels 1 14,161. The synergistic coupling of MIPs with SERS substrates ensures enhanced sensitivity and specificity, making it possible to detect malathion and chlorpyrifos OPPs in complex environmental matrices [12,17]. In addition, AgNPs, due to their excellent optical, localized surface plasmon resonance (LSPR) properties, and thermal and electrical conductivity, have proven to be effective supports for chemical sensing applications [18,19]. Coupling a noble metallic NP, such as silver, with a polymeric platform for sensing pesticide residue significantly enhances the detection of trace pollutants, such as pesticide residues, in the environment [ 12,20-22],

[0082] It is essential to develop robust analytical tools, such as MIP-SERS platforms, for pesticide applications. To advance scientific knowledge, we explored the potential of embedding AgNPs into MIP as active SERS substrates for detecting malathion in drinking and tap water samples. To the best of our knowledge, there is limited information or non-existent work on the in-situ synthesis of silver MIP-SERS substrates for malathion detection. In this study, we developed and characterized a silver MIP SERS substrate and evaluated its potential for detecting malathion. Also, the novelty of coupling MIPs with specific recognition features with SERS substrates is inherent in their ability to synergistically propel the unique signal amplification characteristics of the SERS probe for exceptional sensitivity and selectivity of the target analyte. This MIP-SERS technology has thus become an emerging analytical technique for the distinct detection of target analytes in complex Docket No.: UNCG-532-PCT matrices

[0023] . In addition, MIP-SERS nanosensors offers an innovative approach for enhanced selectivity of analyte detection across a broad-spectrum thus, mitigating the selectivity challenge evident in other analytical sensing platforms

[0024] , This silver MIP-SERS chemosensor could provide insights into rapid analytical tools with promising potential for environmental monitoring applications.

[0083] Herein described, molecularly imprinted polymers (MIPs) embedded with silver nanoparticles (AgNPs@MIPs) were synthesized for the selective detection of malathion by the precipitation polymerization technique using methyl methacrylate (MMA) as a functional monomer, malathion as a template molecule, ethylene glycol dimethacrylate (EGDMA) as cross-linker, 2,2'- azobisisobutyronitrile (AIBN) as an initiator, and the AgNPs as excellent surface-enhanced Raman scattering (SERS) substrates. The platform of AgNPs@MIPs was well characterized regarding its morphology, structure, and spectroscopic features. Our results confirmed that the AgNPs@MlPs specifically recognize malathion from complex matrices such as in drinking and tap water. The intensity of the SERS characteristic peak at 1580 cm1was proportional to the evaluated concentrations, with a linear range of 0.005 to 5 pg / ml and a limit of detection (LOD) of 0.005 pg / ml. The recovery rates and relative standard deviation for the detection of malathion spiked in water samples were ranging from 93% to 100% and from 0.05% to 0.71%, respectively. Overall, the synergistic SERS-MIP sensor method demonstrated superior advantages and novelty in highly selective, sensitive, easy and quick detection of malathion, promising for development of a sensing technology for monitoring many organophosphate compounds in environmental and agriculture applications.

[0084] 1.2 Materials and Methods 1.2.0 Chemicals and reagents

[0085] All chemicals and reagents used were of analytical grade. Malathion peslicide (pestanal analytical), Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), 2,2'- azobisisobutyronitrile (AIBN), acetonitrile (ACN), silver nitrate (AgNO3, > 99%), and sodium borohydride (NaBH4), and were purchased from both Sigma- Aldrich and Fisher Scientific (USA). All purchased chemicals were used as received without further purification. Deionized water (18 M cm) used in this work was acquired from the centralized distillation utility facility at our university.

[0086] 1.2.1 Synthesis of silver molecularly imprinted polymer (AgNPs@MIPs)

[0087] A schematic of the synthesis process and the development of the silver molecularly imprinted polymer is illustrated in FIG. 1.

[0088] After several optimizations of parameters and using a response surface methodology, the best MIP synthesis blend was employed for the development of the AgNPs ©MIP by the precipitation Docket No.: UNCG-532-PCT polymerization technique. Firstly, 38 mg of malathion was mixed with 2 ml of the functional monomer (MAA) and 1.5 ml EGDMA (crosslinker), the mixture was sonicated and homogeneously mixed for 20 minutes in a sample container. 40 mg of AIBN (initiator) and 25 ml of acetonitrile solution containing 400 mg of AgNO ; were added to the mixture and sonicated for another 20 minutes. The chemical structure of the template molecule, initiator, crosslinker, and functional monomer employed in the MIP synthesis is depicted in FIG. 2.

[0089] The homogeneous mixture of all the chemical agents including template malathion and AgNO3 was then transferred to a round bottom flask and thereafter purged with nitrogen gas for 10 minutes. The polymerization of the purged sample was done using an oil bath at a temperature of 65 °C for 12 hours under controlled stirring at 100 rpm. During the polymerization process, the precomplex and the entire mixture gradually solidify. A color change from a transparent to brown coloration occurs after 7 hours till completion of the polymerization reaction. The rigid polymerization was then ground and sieved through a mesh steel sieve (200 p). About 20 g of the malathion-MIP powder doped with AgNOs was then dispersed in 10 ml of 1 mM sodium borohydride (NaBFU) solution for the reduction of the silver precursor into silver nanoparticles (AgNPs). During the addition of the borohydride solution, effervescence occurred from the suspension with the color of the mix transformed to a completely dark brown. The in-situ synthesized AgNPs in the polymer matrix yielded spherically shaped NPs of size ~20 nm (FIG. 3) within the MIP matrix. The solution was filtered with a Whatman filter paper and the malathion-AgNPs@MIP was collected and dried under vacuum overnight till a constant weight was obtained.

[0090] Soxhlet extraction was employed for the removal of the template molecule from the malathion-AgNPs@MIP. In brief, the dried powdered AgNPs @ MIP sample was reacted with 200 ml of methanol / acetic acid (9:1, v / v) for 72 hours, and further reacted with another organic solvent methanol (200 ml) for 24 hours for complete removal of the template malathion.

[0091] Also, a non-imprinted polymer (AgNPs@NIP), which serves as the control was also fabricated with the same technique as the AgNPs @ MIP, however, this sample did not have the malathion as template.

[0092] 1.2.2 Characterization of the silver molecularly imprinted polymer (AgNPs@MIPs)

[0093] The AgNPs @ MIP and the AgNPs @ NIP were both characterized to understand their chemical structure and morphological characteristics. Fourier transform infrared spectroscopy (FTIR) was conducted on the MIP and NIP samples using an Agilent 670 FTIR Spectrometer w / ATR (USA) instrument within a scan range of 4000 to 400 cm1. To confirm the successful doping of AgNPs into the MIP platform, a Rigaku SmartLab X-ray diffractometer (XRD) instrument was used. Also, a Scanning Electron Microscope (SEM) specifically a JEOL JSM-IT800 Schottky FESEM instrument Docket No.: UNCG-532-PCT was used for understanding the morphological features of the synthesized samples. The synthesized AgNPs were also characterized using a TEM instrument (JEOL JEM-2100 plus). UV-Vis absorption spectra were measured on an Agilent Cary 60 instrument (USA) to validate the complete removal of the template molecule from the MIP samples. Raman Spectra were also taken for the samples with a Horiba XploRA Raman Confocal Microscope instrument (USA) with an 1800 grating, a 532 nm laser source, and a 50x long working distance microscope objective. The measurements were performed four times with an average acquisition and accumulation time of 5s with a scan range of 400 cm1to 2500 cm-1.

[0094] 1.2.3 Binding affinity of the silver molecularly imprinted polymer (AgNPs@MIP)

[0095] In evaluating the binding affinity of the fabricated MIP platform, a series of kinetic adsorption tests were conducted to confirm the specific affinity of MIPs@ AgNPs toward malathion. A kinetic adsorption test was conducted by mixing 10 mg of MlPs@ AgNPs and NlPs@ AgNPs with 2 mL of malathion methanol solution (10 pg / ml) at room temperature. The mixture was incubated from 0 to 60 min in the malathion methanol solution at 25 °C. After centrifugation, the supernatant was detected via UV - vis spectroscopy to determine the residual malathion. The adsorption efficiency was then calculated according to the following equation: where Ci is the initial concentration of malathion (mg / L), Cf is the final malathion concentration in the supernatant (mg / L), V is the volume of solution (L), and m (mg) is the dry weight of MIPs@ AgNPs or NIPs@ AgNPs nanocomposites in each adsorption solution.

[0096] 1.2.4 SERS measurements of the silver molecularly imprinted polymer (AgNPs@MIPs) and the AgNPs @ NIP

[0097] The SERS activity of the AgNPs @ MIP and the AgNPs @ NIP substrates were investigated by incubating (30 minutes) with different concentrations of malathion solutions (0.005 pg / ml to 50 pg / ml) collected using a Horiba XploRA Raman Confocal Microscope instrument (Texas, USA) with optimized parameters: 1800 grating, excited at 532 nm, scanning range between 400 cm'1and 2500 cm1, objective lens 50X, laser power 0.5mW, acquisition and accumulation time 5s respectively.

[0098] 1.2.5 Application ofAgNPs@MIP for SERS detection in water samples

[0099] Two different water samples; a bottled drinking water sample purchased from a supermarket in Greensboro, USA, and the municipal tap water sourced from the nanochemistry laboratory at the Joint School of Nanoscience and Nanoengineering, University of North Carolina, Greensboro, USA, were evaluated. Each 10 mg AgNPs @ MIP sample was spiked with 2 ml of the water samples with a concentration of malathion at 20 pg / ml and 1 pg / ml respectively and incubated for 30 minutes. The Docket No.: UNCG-532-PCT

[0100] SERS activity of the AgNPs@MIP was then collected using the optimized Raman parameters (excited at 532 nm, scanning range between 400 and 2500cm- 1, objective lens 50X, laser power 0.5mW, acquisition and accumulation time 5s respectively).

[0101] 1.2.6 Selectivity Test of AgNPs@MIP

[0102] In evaluating the selectivity of the AgNPs@MIP, two other organophosphate pesticides (dimethoate and parathion) were employed. In this instance, a 1 : 1 concentration of malathion and dimethoate each with a concentration of 5 pg / ml was prepared. Similarly, another blend of malathion and parathion with the same concentration as 5 pg / ml respectively was also prepared. 10 mg sample of AgNPs@MIP was each mixed and incubated with 2 ml of the two blends of malathion / dimethoate and malathion / parathion solutions respectively for 30 minutes. The SERS activity of the AgNPs@MIPs was then collected with the same optimized parameters as previously established.

[0103] 1.2.7 Statistical measurements

[0104] For all analyses, the measurement, of each sample was scanned three times, and the average signal was recorded as the UV-Vis, FTIR, and SERS spectra respectively. All spectral data were analyzed in OriginPro 10.05 software (OriginLab Corporation, MA, USA).

[0105] 1.3 Results and Discussions

[0106] 1.3.1 Synthesis and characterization ofAgNPs@MIPs

[0107] In general, for SERS applications, noble metals have proven to be excellent substrates due to their enhanced sensitivity, ease of preparation, and chemical stability

[0025] . Although it is noteworthy that gold nanoparticles possess enhanced chemical stability, their SERS activity is, however, not as strong as noted in silver nanoparticles [26,27], Thus, silver nanoparticles are considered desirable SERS substrates due to their excellent SERS property

[0026] and as such their consideration for the synthesis of the AgNPs@MIP platform.

[0108] The sche malic illustration of the synthesis of AgNPs@MIPs and the SERS detection of malathion was elucidated in FIG. 1. The concept of efficiently doping silver nanoparticles into a polymer matrix or substrate is challenging due to the non-dispersity and non-uniformity of the silver nanoparticles [28,29] within the MIP platform or substrate and subsequently limiting the effectiveness of the MIP substrate as a SERS recognition agent and for analytical detection applications [30,31], To overcome, this challenge, an in-situ synthesis approach can be adopted for the successful doping of uniformly dispersed silver nanoparticles within the MIP platform

[0014] , Thus, a homogenous mixture between the precursor AgNOs, the template molecule (malathion), and the functional monomer methyl acrylic acid results in the even distribution of the silver precursor within the polymer matrix. The reduction of the silver ions by the addition of sodium borohydride results in the formation of AgNPs inside the matrix. Organic solvents play a fundamental role in the functionality of the Docket No.: UNCG-532-PCT fabricated MIP, by ensuring a successful imprinting process when the template has been completely removed [10,12,32], In addition, the template removal creates recognition cavities or activates the imprinted sites in the MIP and the synergistic effect of the SERS active AgNPs within the MIP matrix supports the enhanced and ultrasensitive detection of the target analyte malation

[0010] ,

[0109] The nanostructures and morphologies of the AgNPs @MIPs and AgNPs @NIPs were characterized and evaluated by SEM as elucidated in FIG. 4. The shape of the AgNPs@MIPs and AgNPs @NIPs was oval and had the silver nanoparticles encapsulated on their surface and within the polymer matrix as depicted in FIG. 4. FIG.s 4A and 4B highlight the non-eluted (before) and eluted (after) morphologies of the AgNPs @MIPs respectively as aligned to the malathion template removal process. It was evident that the successful removal of the malathion template with the organic solvent blend of methanol / acetic acid created cavities in the MIP

[0033] and ensured a mesoporous morphological feature

[0034] in the MIP platform thus aclivaling the binding sites of the AgNPs@MIP and its functionality and affinity for a target analyte

[0035] . A similar characteristic observation was also noted in FIG.s 4C and 4D which also highlighted both the non-eluted and eluted AgNPsONIPs respectively, however in the case of the NIP, organic residues are removed from the platform thus creating a porous substrate. Hence, an effective template removal process in the MIP platform creates cavities hence ensuring the binding sites and affinity of the MIP for sensing a target analyte [36,37]. Also, the TEM image as seen from FIG. 3, confirmed the presence of AgNPs doped in the AgNPs @ MIP with an average size of 20 nm. The AgNPs were embedded within the imprinted matrix and on its surface as well with such a densely packed distribution of AgNPs an excellent host of SERS active agents and highly beneficial to support the sensitive SERS detection of malathion.

[0110] 1.3.2 UV-Vis Analysis and AgNPs@MIPs Template Removal Process

[0111] The characterization technique of UV-Vis analysis in the fabrication of MIPs is very critical, as it is an important validation step in confirming the template removal from the MIP platform

[0038] . Also, an observed shift in the UV-Vis spectrum, thus confirms the complete removal of the malathion template from the MIP platform as shown in FIG. 5. Initially as seen in FIG. 5A, the pure malathion is UV-active and depicts a UV-vis spectrum at 252 nm, another spectrum at 262 nm is ascribed to the AgNPs @ MIP before the silver ions were reduced. However, after NaBH4 treatment of the MIP platform, the typical surface plasmon resonance peak around 402 nm was evident confirming the formation of AgNPs within the MIP platform. In addition, FIG. 5B, the UV-Vis spectrum depicting a peak of 406 nm of the eluted AgNPs @ MIP and AgNPs @ NIP, validates and confirms the presence of AgNP doped into the MIP platform after the successful removal of the template malathion thus activating the binding sites of the MIP and increasing its affinity for any reaction mechanism with a target analyte. During the template removal process (FIG. 5C and 5D respectively), a blend of two Docket No.: UNCG-532-PCT organic solvents (methanol / acetic acid) is first employed to remove the malathion from the AgNPs@MIP and depicts a peak around 252 nm. Interestingly, it was observed that the AgNPs@NIP also showed similar characteristics, however, it was confirmed that the pseudo malathion characteristics of the AgNPs@NIP were associated with the functional monomer; methyl acrylic acid as both the MM A and the malathion possessed a similar chemical fingerprint as confirmed by the FTIR analysis of these chemical agents. The final organic solvent employed for the AgNPs@MIP elution process was methanol, which also demonstrated the successful template removal with a shift in the UV-Vis spectrum at a peak of 216 nm. Generally, the disappearance of the UV absorbance peak of the malathion or template molecule, after the solvent elution process confirms, the successful removal of the template molecule from the MIP platform as confirmed by several studies available in the literature [14,23],

[0112] 1.3.3 Adsorption Characteristics ofAgNPs@MIPs

[0113] The development of MIPs with an increased surfaced area and possessing a mesoporous nature are excellent attributes for the efficient and selective rebinding of target analytes such as malathion. Thus, in assessing the rebinding performance of the AgNPs@MIPs, UV-vis spectroscopy was employed to assess the kinetic binding effect of the MIP and the NIP with malathion, as depicted by the adsorption kinetic curves (FIG. 6). The adsorption of malathion for the AgNPs@MIPs reached an equilibrium around 20 mins, it was also however, observed for the AgNPs@NlPs that the time of equilibrium was a little faster than the MIPs, and this could be a result of the interaction of the malathion with the NIPs’ surface which can easily facilitate the diffusion of the malathion. Similarly, the interaction between the AgNPs@NIP and the malathion is attributed to the non-specific binding effect of the malathion to the AgNPs @ NIP whereas, an enhanced significant selective adsorption or specific binding of malathion occurs at the recognition sites inside the AgNPs@MIPs.

[0114] 1.3.4 X-Ray Diffraction (XRD) Analysis of AgNPs@MIPs

[0115] In confirming the presence and the doping of AgNPs into the MIP platform, further characterizations of the AgNPs@MIP were done by X-ray diffraction (XRD) technique. As elucidated in FIG. 7, five predominant diffraction peaks were observed and all were consistent with the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) of the face-centered cubic (fee) of silver nanoparticles, indicating the formation of crystalline silver nanoparticles within the MIP platform. The observed diffraction peaks of silver nanoparticles had diffraction angles of 37.9°, 44.3°, 64.5°, 77.3° and 81.7° within the MIP were confirmed and were consistent with results of other studies [14,39,40] that corroborated the crystallinity of the silver nanoparticles observed in the synthesized AgNPs @ MIP.

[0116] 1.3.5 Fourier Transformed Infrared (FTIR) Analysis of the AgNPs@MIP Docket No.: UNCG-532-PCT

[0117] In the development of MIP substrates, the use of FTIR as a characterization tool is of prime essence, as it helps to distinguish the different chemical bonds that are formed and associated with the core elements of the MIP such as the functional monomer and the template molecule [41,42], Thus, the FTIR analysis confirmed the presence of different functional groups that existed as a result of the interaction between the malathion (template molecule), the functional monomer (MM A), and the cross-linker (EGDMA). FIG. 8 shows the characteristic peaks of the malathion, the MAA, the eluted and non-eluted AgNPs @ MIP as well as the eluted and non-eluted AgNPs @ NIP respectively. It was observed that a strong peak at 1724 cm'1in all evaluated samples (malathion, MAA, NIP, MIP) confirmed the C=O stretching of the ester group in MAA and its interaction with both the cross-linker (EGDMA) and the malathion confirmed the formation of the AgNPs @ MIP [43^-5] . However, in the case of the NIP, the observed peak at 1724 cm1was attributed only to the C=O stretching of the ester group in the MAA [23,46]. Also, another strong peak at 1140 cm1observed in the MAA, MIP, and the NIP could be attributed to the stretch from the C=O ether group in the MAA. Similarly, the observed peak of 1140 cm'1in the MIP is closer to the peak at 1158 cm'1confirmed in the malathion which could confirm the interaction of the MIP with the malathion template. Also, the peaks in the range of 500 - 1000 cm1in the malathion spectrum are all attributed to the vibrational stretching of the functional groups (P=S, P-O-C, and the P-O) [47,48],

[0118] It was also observed that a medium peak at 2950 cm1which was prominent in all the spectrum may indicate C-H stretching of the alkyl group in both the pure malathion and the functional monomer (MAA). Thus, in the case of the MIP, the observed C-H stretch at 2950 cm'1is a result of the interaction between the alkyl group in the MAA, and malathion with the EGDMA

[0049] , However, the similar observed peak in the NIP may be due to the C-H stretch of the alkyl group as a result of its interaction with the EGDMA. Also, the broad peak at 3500 cm1observed in the non-eluted MIP may be due to the O-H stretch in the carboxylic group in the MAA

[0049] , however, this peak was lost after the template was removed by the solvent elution process.

[0119] Also, FIG. 9, confirms the characteristic similarities of the FTIR analysis between the AgNPs @ MIP and the AgNPs @ NIP. It can be significantly confirmed that the strong peak at 1724 cm'1confirms the C=O stretch in the carboxylic group in the MAA and its interaction with the crosslinker EGDMA. Also, the observed peak at 1140 cm'1is indicative of the C-0 stretch of the carboxylic acid group in the MAA. The other medium peaks confirmed at 750 cm'1, 985 cm'1,and 1452 cm'1are linked to the C-H, and C-0 stretches in the MAA and its interaction with the EGDMA. Also, the peak around 2950 cm'1is a result of the C-H stretch in the MAA, and its interaction with the EGDMA.

[0120] 1.3.6 Surface Enhanced Raman Spectroscopy (SERS) Analysis of AgNPs@MIPs Docket No.: UNCG-532-PCT

[0121] SERS is an important non-invasive ultra-sensitive technique that has the potential to detect a broad spectrum of analytes including malathion. The incorporation of SERS substrates into MIP platforms has great analytical detection potential, as it employs signal amplification by plasmonic or chemical effects from Raman active molecules such as from silver nanoparticles which is a good SERS host. This amplified signal therefore depicts a specific vibrational Raman spectrum which connotes the fingerprint of the target analyte when there is a binding event

[0012] , Thus, the mechanism of SERS was studied by the rebinding of malathion with the AgNPs@MIP and the AgNPs@NIP as elucidated in FIG. 10. The observed Raman shift of the binding of the analyte with the eluted MIP substrate was confirmed within the range of 500 - 2500 cm1. The pure malathion standard depicted significant Raman bands within the range of 500 -1600 cm1which corresponded to the vibration of molecules from the malathion template molecule, hence the vibration of the malathion characteristic bands within this range could be ascribed or assigned to the stretching of the P=S, P-O-C, and the P-0 bonds within the compound. Also, the Raman bands of the AgNPs@MIP were significantly pronounced within the platform with the doped silver nanoparticles enhancing the SERS signal amplification. The observed characteristic bands are evidence of the interaction between the silver nanoparticles in conjunction with the malathion imprinted polymer. It was also evident that a characteristic fingerprint and peak region between 1500 cm1and 1600 cm1had intense SERS amplification and was related to the electromagnetic enhancement of the Ag substrate [ 141 within the MIP. This characteristic fingerprint in the MIP was also substantiated to be silver nanoparticles as confirmed by the Raman scattering spectra of synthesized silver nanoparticles in a study by Joshi et al.

[0050] , This fingerprint region thus, became a signature peak for the AgNPs@MIP’s rebinding event with the malathion template and specifically showed a significantly enhanced Raman peak at 1580 cm1which was evident in all samples reacted with the malathion solution.

[0122] On the contrary, not many Raman bands were observed for the AgNPs @ NIP except for the signature peak of the silver nanoparticles that showed SERS enhancement between 1500 cm'1and 1600 cm1. This observation was expected as the polymers were non-imprinted and there was limited interaction between the AgNPs and the NIP platform due to the weak adsorption and non-specific recognition of the analyte

[0023] , On the contrary, after rebinding the AgNPs@NIP with 10 pg / ml, the Raman bands were slightly pronounced as compared to the control AgNPs @ NIP and this could be attributed to the SERS enhancement of the AgNPs @ NIP reacting with the malathion solution. Interestingly, when there was a rebinding event of the eluted malathion AgNPs @ MIP with the same concentration of malathion (10 JJ g / inl ), the signature peak depicted more SERS enhancement at 1580 cm1which was previously confirmed in FIG. 10. It was also worth noting that, after the rebinding event of the AgNPs@MIP with the malathion solution, the Raman bands between 500 cm1to 1000 Docket No.: UNCG-532-PCT cm1were all remodeled and depicted an umbrella-like figure signifying the strong specificrecognition and hydrogen bonding and electrostatic interaction of the AgNPs@MIP with the malathion

[0023] ,

[0123] 1.3.7 SERS Assay and Binding of AgNPs@MIP with Malathion Concentrations

[0124] The binding of different concentrations of malathion solutions from the range of 0.005 pg / ml to 50 pg / ml was evaluated for the SERS activity of AgNPs@MIP as depicted in FIG. 11. The characteristic SERS peak at 1580 cm1was prominent in all the spectra with a strong enhancement when the highest concentration of malathion (50 pg / ml) was bound to the AgNPs@MIP. The incubation time of about 30 minutes was also highly efficient in ensuring the equilibrium of the AgNPs @ MIP in adsorbing and enriching the target analyte into the MIP’s matrix for effective detection. Consequently, the excellent SERS spectra as showcased in FIG. 11, confirm the selective potential of MIPs in detecting a wide range of concentrations of specific target analytes. Also, the densely oriented AgNPs in the matrix of the MIP essentially supported the electromagnetic field enhancement which resulted in an intense and superior SERS signal for the sensitive detection of the malathion analyte [27,51], It was observed and confirmed that the characteristic SERS peak at 1580 cm1was enhanced from FIG. 11c to 11g, which corresponded to the increasing malathion concentrations. It can thus be inferred that the SERS enhancement was directly proportional to the malathion concentrations. Also, the mesoporous structure of the AgNPs@MIP aided its efficiency in adsorbing the target analyte. It was also confirmed that the highest SERS intensity (5000) was reported for the AgNPs @ MIP that was incubated with 50 pg / ml of malathion solution and this intensity was enhanced by the SERS substrate embedded in the AgNPs @ MIP. These results confirm the promising potential of the MIP and its sensitivity for the target analyte malathion. The successful analytical SERS detection of malathion was thus confirmed by the binding event of the incubated AgNPs@MIP with malathion solutions at concentrations of 0.005 pg / ml (c), 0.05 pg / ml (d), 0.5 pg / ml (e), 5 pg / ml (f), and 50 pg / ml (g) respectively, with an enhanced SERS intensity band of malathion detected at 1580 cm1by the AgNPs® MIP.

[0125] 1.3.8 Linearity of AgNPs@MIP SERS for malathion detection

[0126] The linearity of the SERS method for malathion detection was evaluated by taking four standard calibration points against the characteristic peak intensity at 1580 cm1for concentrations from 5 pg / ml to 0.005 pg / ml. The Raman band at 1580 cm1which is assigned to the vibrational stretch of the electrostatic interaction between the AgNPs and the P=O and P-0 groups in the malathion was considered due to its distinct and strong intensity as compared to other bands in the spectrum. Also, the logarithmic transformation of data with a wide range of dispersion has proven to be very useful specifically in SERS calibration curves [52-54], Thus, the exhibited concentration and Docket No.: UNCG-532-PCT peak area intensity in logarithmic yielded a linear response for the evaluated concentrations as elucidated in FIG. 12. The logarithm (log C) of the malathion concentrations had a good linear relationship with the logarithm (log A) of the Raman peak area intensity at 1580 cm1. The linear equation was log A = 2.60445 + 0.73673 x log C (R2= 0.99367). In general, the rule of thumb for the standard minimum detection limit is based on this, when the ratio of the signal intensity (S) to noise intensity (N) of the tested sample is greater than or equal to 3, it is considered to be an effective signal. Therefore, the minimum detection limit of this novel SERS method was reported as 0.005 pg / ml.

[0127] 1.3.9 Selectivity ofAgNPs@MIP

[0128] The selectivity test of the fabricated AgNPs@MIP was evaluated to determine the SERS sensing capability of the MIP for potential OPP target analytes. In this instance, two pesticides namely, dimethoate and parathion were evaluated separately and as a blend with malathion solution with a concentration of 5 prg / nrl respectively as highlighted in FIG. 13.

[0129] As observed from FIG. 13A, the characteristic SERS peak of 1580 cm'1was confirmed after incubating both the malathion together with the dimethoate solution at the same concentration of 5 pg / ml. Similarly, it was also realized that the characteristic SERS peak was also confirmed at 1580 cm1when the AgNPs@MIP was incubated with the dimethoate solution. This development with the dimethoate OPP sharing a SERS characteristic feature with the malathion OPP could be attributed to the aliphatic

[0055] nature of these two compounds, thus the dimethoate possesses a unique ability to act like the malathion when present in a sample. As such, the blend of malathion and dimethoate depicted a narrower characteristic SERS peak at 1580 cm1. It can thus, be confirmed that the AgNPs@MIP has a selective feature for malathion and may also detect other aliphatic OPPs if present in any complex matrix as well, which makes it a promising feature of the fabricated AgNPs@ MIP.

[0130] Also, from FIG. 13B, which had the blend of malathion and parathion at the same concentration (5 pg / ml), it was observed that when the AgNPs@MIP was incubated with the parathion solution only, no characteristic SERS peak was observed at 1580 cm1. This thus inferred that there was no affinity or binding event at the sites of the AgNPs@MIP since there was no recognition of the target analyte parathion in the MIP since the parathion possessed aromatic domains completely different from that of malathion, an aliphatic OPP

[0056] , However, when the blend of malathion and parathion was incubated with the AgNPs@MIP, the characteristic SERS peak at 1580 cm'1was observed. This observation is largely due to the binding sites of the AgNPs@MIP reacting to and possessing a strong affinity towards the malathion solution. In addition, since the parathion served as an interference for the malathion, within the OPP blend, it was observed that the SERS intensity was moderately enhanced as compared to the SERS intensity observed in FIG. 13A. Docket No.: UNCG-532-PCT

[0131] 1.3.10 Real Sample Application of AgNPs@MIP and the Detection of malathion spiked in Selectivity of AgNPs@MIP

[0132] To better understand, the accuracy of the SERS sensing performance of the AgNPs@MIP, evaluating with real samples such as a spiked bottled drinking water and tap water samples with 20 pg / ml and 1 pg / ml of malathion concentration was necessary as confirmed in FIG. 14A and FIG. 14B respectively. The successful detection of malathion in spiked drinking water was confirmed as observed in FIG. 14A. The characteristic SERS peak at 1580 cm1was confirmed with an enhanced SERS intensity, thus, a higher spiked concentration yielded a higher SERS intensity.

[0133] In addition, it was observed that the less spiked concentration of drinking water (1 pg / ml), also depicted the unique SERS intensity peak at 1580 cm1, however, the range of the Raman shift between 500 cm1and 1000 cm1had other sensitive Raman bands highlighted during the SERS enhancement.

[0134] For the tap water sample as well, the unique characteristic SERS peak intensity at 1580 cm‘ was confirmed too. It was thus, also significant to know that the SERS intensity was directly proportional to the spiked tap water concentrations with 20 pg / ml malathion sample exhibiting an intense SERS Raman band. Ultimately, the cavities in the MIP platform had an enhanced binding potential in the presence of the analyte malathion and was able to effectively detect malathion in the water sample. Another factor that could also support the enhanced SERS detection by the AgNPs@MIP in both the drinking and tap water is the phosphorus mineral content of these samples

[0057] , It is mostly known that tap water contains a higher dose of phosphorus due to the addition of orthophosphate as a water treatment process

[0058] , while drinking water contains a moderate amount of phosphate largely due to chemical softeners that are employed

[0059] , It is also worth noting that the AgNPs@MIP had excellent recoveries as highlighted in Table 1.

[0135] Table 1 Detection and removal of malathion from environmental samples

[0136] Sample Spiked Recovered Recovery (%) Standard Relative Standard

[0137] (pg / ml) (pg / ml) Deviation Deviation (%)

[0138] Drinking Water 1 0.93 93.00 0.05 0.05

[0139] Drinking Water 20 19.88 99.40 0.09 0.43

[0140] Tap Water 1 0.99 98.00 0.01 0.71

[0141] Tap Water 20 20.10 100.50 0.07 0.35

[0142] 1.3. Conclusion Docket No.: UNCG-532-PCT

[0143] An enhanced and efficient AgNPs@MIP SERS sensor was produced by the successful doping of AgNPs within the MIP matrix via an in- situ technique for the sensitive and selective detection of malathion, an OPP. The MIP SERS sensor had promising results with a limit of detection of 0.005, as well as showed an enhanced SERS intensity with a linear relationship with concentrations of malathion detected by the AgNPs @ MIP in the range from 50 pg / ml to 0.005 pg / ml. The AgNPs @ MIPs also depicted an excellent SERS selectivity and performance when tested with real samples such as spiked drinking and tap water at concentrations of 20 pg / ml and 1 pg / ml respectively and yielded great recovery rates as well. This novel malathion AgNPs@MIP sensor has an excellent potential for broad spectrum environmental monitoring applications and could be recommended for analytical detection of environmental contaminants in complex matrices.

[0144] Example 2: A One-Pot Synthesis of a Molecularly Imprinted Sensor for Dimethoate Pesticide Detection based on Surface-Enhanced Raman (SERS) Spectroscopy

[0145] 2.1 Introduction

[0146] Pest and disease proliferation in the agricultural sector continues to hamper yield and productivity for many agricultural stakeholders globally and specifically in developing counties [60,61], Evidence-based cases have demonstrated that pesticides do impact food security by boosting crop yield [62,63]. Although pesticides have become a necessary evil to combat pests and diseases, their excessive use disrupts the natural balance of the ecosystem, contaminates food and water bodies, and also poses a threat to human health

[0064] , Dimethoate, an important organophosphate pesticide, plays a pivotal role in addressing the challenges of agricultural pests by inhibiting acetylcholinesterase activity. Despite, the beneficial impact of dimethoate pesticide, residues from this pesticide when in contact with any material such as food have serious health implications, cancer- inclusive for human and public health safety. Dimethoate residues also pose a negative risk to the ecosystem when they are exposed to the air, soil, or water bodies

[0065] .

[0147] Moreover, as pesticide residues have become a major trade barrier for agricultural produce, there is a need to adopt analytical technologies that can adequately determine the level of pesticide residue contamination [66,67], Classically, the gold standard for pesticide analytical detection involves official techniques such as thin-layer chromatography, gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC) [68,69], Despite the precision and sensitivity of these notable pesticide analytical detection techniques, limitations do exist in their use especially for in-field applications, as these instruments can only perform analytical detection in a laboratory environment

[0070] . In addition, their expensive nature, and inherent complexity, require trained technical staff for their operation as well as coupled with time-consuming Docket No.: UNCG-532-PCT analyses [65,70,71]. Thus, the development of a rapid, non-complex, and sensitive technique of dimethoate pesticide analysis that has the potential for trace-level detection and in-field applications is of great interest and this could be achieved by employing molecularly imprinted polymers (MIPs). MIP is a highly efficient and versatile polymeric material with binding sites and cavities complementary to the target analyte which is used as the template molecule

[0010] , Primarily, MIPs, are produced via a polymerization reaction between a functional monomer, and a template molecule in the presence of an appropriate cross-linker, initiator, and porogen (solvent) and have been efficiently employed for the trace-level detection of environmental contaminants [10,12,45],

[0148] Also, spectroscopic techniques, particularly surface-enhanced Raman spectroscopy (SERS), a powerful tool, have been employed for pesticide detection due to its rapid and non-destructive feature and its enhanced sensitivity for trace pesticide detection [64,72,73], SERS is a versatile and distinct technique with the main mechanism of action based on the enhancement of the Raman spectral signal intensity of the target analyte especially, when the target analyte is in proximity to metallic nanostructured surfaces such as silver (Ag) and gold nanoparticles (AuNPs) which are excellent SERS substrates [25,74],

[0149] The development of rapid and sensitive analytical detection techniques is very vital in many fields, especially for the monitoring and detection of environmental contaminants. Molecularly imprinted polymer (MIP) surface-enhanced Raman spectroscopy (SERS) sensors have gained significant attention recently for the trace detection of target analytes.

[0150] Thus, the synergistic coupling of MIPs and SERS substrates such as silver nanoparticles, opens an avenue for a robust and sensitive analytical technique of pesticide detection at the trace level. Moreover, silver nanoparticles (AgNPs) have excellent optical and localized surface plasmon resonance characteristics and hence will efficiently support the enhanced Raman signal for pesticide detection [75,76], As such, this technology has promising potential for environmental contaminant detection at very low concentrations.

[0151] Therefore, in this study, we explored the synthesis of a dimethoate AgNPs ©MIP-SERS substrate and evaluated its potential for the detection of dimethoate in complex environmental matrices such as orange and apple juices. Our study employed an innovative in-situ synthesis concept of embedding spherically shaped silver nanoparticles in the MIP platform which depicts our novelty in this synergistic chemosensing platform for dimethoate pesticide detection. Moreover, to the best of our knowledge, there is little or non-existent work on this concept for dimethoate pesticide detection. Also, this novel AgNPs ©MIP-SERS platform has enhanced selectivity and sensitivity for dimethoate detection, it could also mitigate enhanced detection challenges, possesses broad-spectrum applications, and consequently, could be employed for enviromnental contaminant monitoring. Docket No.: UNCG-532-PCT

[0152] In our study, we developed a MIP-SERS sensor by the in-situ embedment of silver nanoparticles (AgNPs) within a MIP matrix for the sensitive and selective detection of dimethoate, an organophosphate pesticide via a precipitate polymerization technique. The in-situ embedded AgNPs within the MIP matrix offer a homogeneous distribution of SERS hot spots providing superior interaction and affinity between the AgNPs and the target analyte dimethoate. Our AgNPs @ MIP- SERS sensor showed enhanced sensitivity for dimethoate with superior selectivity against other pesticides such as chlorpyrifos and fenthion. The fabricated AgNPs @ MIP also possessed superior selectivity for dimethoate as confirmed in complex matrices such as apple and orange juices spiked with the target analyte. The intensity of the SERS characteristic peak at 1587 cm1was proportional to the evaluated concentrations, with a linear range of 0.005 to 5 pg / ml and a limit of detection (LOD) of 0.005 pg / ml. The recovery rates and relative standard deviation for the detection of dimethoate spiked in the food samples ranged from 94% to 102% and from 0.85% to 4.37%, respectively. Our one-pot synthesized novel chemical nanosensor thus, demonstrated reliable results and has promising potential for practical applications such as for the rapid environmental monitoring of contaminants in complex matrices.

[0153] 2.2 Materials and Methods 2.2.0 Chemicals and reagents

[0154] All chemicals and reagents used were of analytical grade. Dimethoate pesticide (pestanal analytical), Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), 2,2'- azobisisobutyronitrile (AIBN), acetonitrile, silver nitrate (AgNO3, > 99%), and sodium borohydride (NaBH4), and were purchased from both Sigma-Aldrich and Fisher Scientific (USA). All purchased chemicals were used as received without further purification. Deionized water (18 MQ cm) used in this work was acquired from the centralized distillation utility facility at our university.

[0155] 2.2.1 Synthesis of the dimethoate silver molecularly imprinted polymer (AgNPs@MIPs)

[0156] A schematic of the synthesis process and the development of the silver molecularly imprinted polymer is illustrated in FIG. 15.

[0157] The precipitation polymerization technique was employed for the synthesis of the AgNPs @ MIP and the NIP samples respectively by using optimized parameters based on response surface methodology. Briefly, 4 mmol of the functional monomer (MMA) was mixed with template molecule dimethoate (0.22 mmol), and 20 mmol EGDMA (crosslinker), the mixture was vortexed and homogeneously mixed for 20 minutes in a sample container. 0.55mmol AIBN (initiator) and 30 ml of acetonitrile solution containing 500 mg of AgNCh were added to the mixture and vortexed for 20 minutes for complete dissolution of all the chemical components. The chemical struc ure of the Docket No.: UNCG-532-PCT template molecule, initiator, crosslinker, and functional monomer employed in the MIP synthesis is depicted in FIG. 16.

[0158] The homogeneous mixture of all the chemical agents was then transferred to a round bottom flask and thereafter purged with nitrogen gas for 10 minutes. The polymerization of the purged sample was done using an oil bath at a temperature of 70°C for 12 hours under controlled stirring at 100 rpm. During the polymerization process, the pre-complex and the entire mixture gradually solidify. A color change from a transparent to brown coloration occurs after 7 hours till completion of the polymerization reaction. The rigid polymerization was then ground and sieved through a mesh steel sieve (300p). About 20 g of the dimethoate MIP powder doped with AgNOi was then dispersed in 10 ml of 1 M sodium borohydride (NaBFU) solution for the reduction of the silver precursor into silver nanoparticles (AgNPs). During the addition of the borohydride solution, effervescence occurred from the suspension with the color of the mix transformed to a completely dark brown. The in-situ synthesized AgNPs in the polymer matrix yielded spherically shaped nanoparticles of size, 20 nm (FIG. 17) within the MIP matrix. The solution was filtered with a Whatman filter paper and the dimethoate AgMIP was collected and dried under vacuum overnight till a constant weight was obtained.

[0159] Soxhlet extraction was employed for the removal of the template molecule from the AgMIP. The dried powdered AgNPs @ MIP sample was reacted with 200 ml of methanol / acetic acid (9: 1, v / v) for 72 hours, and further reacted with another organic solvent methanol (200 ml) for 24 hours for complete removal of the template. Validation of the template removal process was confirmed with a Cary 60 UV-Vis spectrometer instrument thus confirming the complete removal of the dimethoate template from the AgNPs @ MIP platform.

[0160] Also, a non-imprinted polymer (AgNIP), which served as the control was also fabricated under the same conditions as the AgMIP, however, this sample did not have the dimethoate template. 2.2.2 Characterization of the dimethoate silver molecularly imprinted polymer (AgNPs@MIPs)

[0161] The AgNPs @ MIP and the AgNPs @ NIP were both characterized to understand their chemical structure and morphological characteristics. Fourier transform infrared spectroscopy (FTIR) was conducted on the MIP and NIP samples using an Agilent 670 FTIR Spectrometer w / ATR (USA) instrument within a scan range of 4000 to 400 cm1. In confirming the successful doping of silver nanoparticles into the MIP platform, a Rigaku SmartLab X-ray diffractometer (XRD) instrument was used. Also, a Scanning Electron Microscope (SEM) specifically a .TEOL JSM-IT800 Schottky FESEM instrument aided in understanding the morphological features of the synthesized samples. The synthesized AgNP was also characterized using a TEM instrument (IEOL JEM-2100 plus). Similarly, UV-Vis spectra analyses were measured on an Agilent Cary 60 instrument (USA) to validate the Docket No.: UNCG-532-PCT complete removal of the template molecule from the MIP sample. Raman Spectra were also taken for the samples with a Horiba XploRA Raman Confocal Microscope instrument (USA) with an 1800 grating, a 532 nm laser source, and a 50X long working distance microscope objective. The measurements were performed four times with an average acquisition and accumulation time of 5s with a scan range of 400 cm1to 2500 cm1.

[0162] 2.2.3 The binding affinity of the dimethoate silver molecularly imprinted polymer (AgNPs@MIP)

[0163] In evaluating the binding affinity of the fabricated MIP platform, a series of kinetic adsorption tests were conducted to confirm the specific affinity of MIPs@AgNPs toward dimethoate. A kinetic adsorption test was conducted by mixing 10 mg of MIPs@ AgNPs and NIPs@ AgNPs with 2 mL of dimethoate methanol solution (10 pg / ml) at room temperature. The mixture was incubated from 0 to 60 min in the dimethoate methanol solution at 25 °C. After centrifugation, the supernatant was detected via UV - vis spectroscopy to determine the residual dimethoate. The adsorption efficiency was then calculated according to the following equation:

[0164] Where Ci is the initial concentration of dimethoate (mg / L), Cf is the final dimethoate concentration in the supernatant (mg / L), V is the volume of solution (L), and m (mg) is the dry weight of MIPs@AgNPs or NIPs@ AgNPs nanocomposites in each adsorption solution.

[0165] 2.2.4 SERS measurements of dimethoate silver molecularly imprinted polymer (AgNPs@MIP) and AgNPs @NIP

[0166] The SERS activity of the AgNPs @ MIP and the AgNPs @ NIP substrates were investigated by incubating (25 minutes) the MIP and NIP samples with different concentrations of dimethoate solutions (0.005 pg / ml to 20 pg / ml). SERS measurements were done using a Horiba XploRA Raman Confocal Microscope instrument (Texas, USA) with optimized parameters: 1800 grating, excited at 532 nm, scanning range between 400 cm'1and 2500 cm'1, objective lens 50X, laser power 0.5mW, acquisition and accumulation time 5s respectively.

[0167] 2.2.5 Application of AgNPs@MIP for SERS detection in food samples (Apple and Orange Juice)

[0168] Two different food samples: apples and oranges were purchased from a local grocery store in Greensboro, USA, and were pulverized, and their extracts (apple and orange juice respectively were used for the analysis). Each 10 mg AgNPs@MIP sample was spiked with 2 ml of the apple and orange juice (non-filtered) samples with a concentration of dimethoate at 10 pg / ml and 0.5 pg / ml respectively and incubated for 30 minutes. The SERS activity of the AgNPs@MIP was then collected using the optimized Raman parameters (excited at 532 nm, scanning range between 400 and 2500cm- 1, objective lens 50X, laser power 0.5mW, acquisition and accumulation time 5s respectively). Docket No.: UNCG-532-PCT

[0169] 2.2.6 Selectivity Test of Dimethoate AgNPs@MIP

[0170] In evaluating the selectivity of the AgNPs@MIP, two other organophosphate pesticides (Chlorpyrifos and fenthion) were employed. In this instance, a 1 : 1 concentration of dimethoate and chlorpyrifos each with a concentration of 5 jig / ml was prepared. Similarly, another blend of dimethoate and fenthion with a specific concentration of 5 pg / ml was also prepared. 10 mg sample of AgNPs@MIP was each mixed and incubated with 2 ml of the two blends of dimethoate / chlorpyrifos and dimethoate / fenthion solutions respectively for 20 minutes. The measurement of their SERS activity was then evaluated.

[0171] 2.2.7 Statistical measurements

[0172] For all analyses, the measurement, of each sample was scanned three times, and the average signal was recorded as the UV-Vis, FTIR, and SERS spectra respectively. All spectral data were analyzed in OriginPro 10.05 software (OriginLab Corporation, MA, USA).

[0173] 2.3 Results and Discussions

[0174] 2.3.1 Synthesis and characterization of dimethoate AgNPs@MIP

[0175] The coupling of metallic nanoparticles such as silver nanoparticles with polymeric materials has attracted widespread attention as a result of their synergistic effect and multifunctional properties including their potential to be adopted as excellent SERS substrates for a myriad of applications

[0077] , AgNPs are thus, recognized as an excellent SERS substrate, and most frequently employed in optical field detection due to their distinct optical properties. In addition, AgNPs have a more robust and efficient SERS enhancement than AuNPs as a result of their potential to exhibit a strong surface electric field under plasmonic excitation [77,78], as such their selection for the fabrication of the dimethoate AgNPs ©MIP. The schematic illustration of the fabrication of the dimethoate AgNPs @MIP is depicted in FIG. 15.

[0176] Doping AgNPs in a polymeric matrix or substrate is an arduous task, owing to the nonuniformity and non-dispersity of the AgNPs [28,29] within the MIP substrate thus, impeding the MIP- SERS’s performance [30,31], In addressing, this challenge, an in-situ synthesis approach can be adopted for the successful embedding of uniformly dispersed AgNPs within the MIP substrate

[0014] , Thus, a homogenous blend and reaction of dimethoate (template molecule) the precursor (AgNOs), and methyl acrylic acid (functional monomer) results in the uniform distribution of the precursor AgNOs within the polymeric substrate. Consequently, the addition of sodium borohydride reduces the silver ions present in the polymer to form spherically shaped AgNPs inside the polymer matrix. Also, organic solvents such as acetonitrile play a vital role in the MIP’s functionality, by ensuring a successful imprinting process when the template has been completely removed [10,12,32], In addition, the removal of the template from the host polymer creates recognition cavities and activates Docket No.: UNCG-532-PCT the binding potential of the imprinted sites in the MIP. Furthermore, the AgNPs within the MIP matrix support the superior detection of the dimethoate analyte

[0010] .

[0177] The nanostructures and morphologies of the AgNPs @ MIP and AgNPs @ NIP were characterized and evaluated by SEM as elucidated in FIG. 18. FIG. 18 (A and B) highlight the imprinted dimethoate Ag substrate and the non-imprinted Ag substrate respectively. It was evident that the successful removal of the dimethoate template with the organic solvent blend of methanol / acetic acid created cavities in the MIP

[0033] and ensured a mesoporous morphological feature

[0034] in the MIP substrate thus activating the binding sites of the AgNPs@MIP

[0035] . However, in the case of the NIP, organic residues are removed from the platform thus creating a porous substrate. Hence, an effective template removal process in the MIP platform creates cavities hence ensuring the binding sites and affinity of the MIP for sensing target analytes [36,37], Also, the TEM image as seen from FIG. 17, confirmed the presence of AgNPs doped in the AgNPs@MlP with an average size of 20 nm. The AgNPs were embedded within the imprinted matrix and on its surface as well thus creating excellent SERS hot spots to support the sensitive SERS detection of dimethoate.

[0178] 2.3.2 Dimethoate AgNPs@MIP: Template Removal Process and Validation with UV-Vis Analysis

[0179] UV-Vis analysis plays a significant role in the design and synthesis of MIP substrates as it supports in confirming and validating the template removal from the MIP platform

[0038] . FIG. 19 (A), highlights the pure dimethoate as a UV-active molecule and depicts a UV-vis spectrum at 245 nm, also, the AgNPs depict a spectrum at 408 nm in the eluted AgNPs@MIP after the sample is eluted. FIG. 19 (B), also confirms the complete removal of the dimethoate template from the MIP platform with the two different organic solvents consisting of a blend of methanol (9 parts) to 1 part of acetic acid. It was observed that there was a shift, in the UV-Vis spectrum (214 nm), after methanol only was used for the final elution step thus confirming the removal of dimethoate from the AgNPs @ MIP. In principle, the loss of the UV-absorbance peak of the dimethoate molecule, after the solvent elution process confirms, the successful removal of the template molecule from the MIP platform as evidenced by several studies in the literature [14,23],

[0180] 2.3.3 Adsorption Characteristics of Dimethoate AgNPs@MIP

[0181] MIPs with mesoporous surface characteristics adequately support the rebinding of target analytes

[0079] such as dimethoate thus, the rebinding potential of the dimethoate AgNPs @ MIP was assessed with UV-vis spectroscopy to determine the kinetic binding effect of the MIP and the NIP (FIG. 20). The adsorption of dimethoate for the AgNPs @ MIPs reached an equilibrium around 30 mins, and gradually leveled up, with a similar trend also observed in the case of the AgNPs @NIPs. It was however, evident that the interaction between the dimethoate analyte and the AgNPs @ NIP was Docket No.: UNCG-532-PCT aligned to the non-specific binding affinity of the AgNPs@NIP to the dimethoate whereas, the significant selective binding adsorption or of dimethoate occurred at the AgNPs@MIP’s binding sites.

[0182] 2.3.4 X-Ray Diffraction (XRD) Analysis of AgNPs@MIP

[0183] In validating the successful doping and integration of AgNPs into the dimethoate AgNPs@MIP substrate, X-ray diffraction (XRD), was employed for such characterization (FIG. 21). Five distinct silver nanoparticles diffraction peaks were observed with high crystallinity and were all consistent with the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) of the face -centered cubic (fee) of silver nanoparticles within the MIP platform. Thus, the observed diffraction peaks of silver nanoparticles within the MIP had diffraction angles of 37.9°, 44.3°, 64.5°, 77.3° and 81.7° were confirmed and consistent with the available literature results of other studies [14,39,40],

[0184] 2.3.5 Fourier Transformed Infrared (FTIR) Analysis of AgNPs@MIP

[0185] FTIR is a distinct tool that highlights the intricate chemical interaction and bonds formed between the dimethoate template molecule, the methyl acrylic acid functional monomer and EGDMA (cross-linker) as observed in the synthesized dimethoate AgNPs @ MIP. This chemical bond formation between the core elements of the fabricated MIP is distinguished by the presence of different functional groups [10,80-82], In our study, we observed characteristic FTIR peaks (FIG. 22) of the dimethoate template molecule, the MAA, and the eluted AgNPs@MIP as well as the eluted AgNPs@NlP respectively. A strong peak at 1723 cm1in the characterized samples (dimethoate, MAA, MIP and NIP) represents the C=O stretching of the ester group in MAA and its interaction with both the cross-linker (EGDMA) and the dimethoate confirmed the formation of the AgNPs @ MIP [43^-5]. Interestingly, the observed peak at 1723 cm1in the NIP was attributed only to the C=O stretching of the ester group in the MAA [23,46], Also, a notable peak at 1140 cm1reported in the NIP, MIP, and the MAA, is attributed to the stretching of the C=O ether group in the MAA. In addition, the observed peak of 1639 cm1in the dimethoate confirmed the amine (NH-CO) functional group in the dimethoate template molecule

[0083] . Also, the peaks in the range of 500 - 1002 cm'1in the dimethoate spectrum are all attributed to the vibrational stretching of the functional groups (P-S, C-S, and P-O-C,) [84,85], It was also observed that a medium peak at 2956 cm'1as confirmed in FIG. 22 indicated the C-H stretching of the alkyl group in both the pure dimethoate and the functional monomer (MAA). Consequently, the observed C-H stretch at 2956 cm'1in the MIP is indicative of the chemical interaction between the alkyl group in the MAA, and dimethoate with the EGDMA

[0049] . However, the similar observed peak in the NIP may be due to the C-H stretch of the alkyl group as a result of the MAA’s reaction with the EGDMA.

[0186] Also, FIG. 23 and FIG. 24, confirm characteristic FTIR bands when the synthesized dimethoate AgNPs @ MIP and AgNPs @ NIP were treated with 5 pg / ml and 20 pg / ml dimethoate Docket No.: UNCG-532-PCT solution respectively. It was evident that the AgNPs@MIP had a stronger binding affinity for the dimethoate solution than the AgNPs@NIP substrate, as new FTIR peaks (1024 cm1and 2834 cm1) were observed as a result of the binding affinity of the MIP towards the dimethoate solution. The observed new FTIR peaks in the AgNPs@MIP were 1024 cm1and 2834 cm1corresponding to the P- O-C and C-H functional groups respectively. Several studies have also corroborated the appearance of new FTIR bands when MIP substrates are treated with their target analytes. This is largely due to the enhanced affinity of the MIP for its recognition element [86-88]. It can also be significantly reported that the strong peak at 1725 cm'1confirms the C=O stretch in the carboxylic group in the MAA and its interaction with the cross-linker EGDMA. Also, the observed peak at 1147 cm'1is indicative of the phosphoryl (P=O) stretch in the dimethoate molecule. Also, the peak around 2950 cm'1is a result of the C-H stretch in the MAA, and its interaction with the EGDMA.

[0187] In addition, the FUR peaks around 3500 cm'1observed in both the MIP and NIP samples after adsorption with the dimethoate solution confirmed the O-H functional groups as a result of the MAA’s interactive force in the polymeric substrates (MIP and NIP) as confirmed and reported by other studies [89,90],

[0188] 2.3.6 Surface Enhanced Raman Spectroscopy (SERS) Analysis of AgNPs@MIP

[0189] SERS is an ultra-sensitive and vital analytical technique for the trace detection of analytes such as dimethoate pesticides. The synergistic effect of coupling MIP matrices with SERS substrates such as silver nanoparticles has enhanced signal amplification of the target analyte due to inherent chemical or plasmonic properties associated with Raman active molecules (silver nanoparticles) [12,91,92], Also, in the presence of a target analyte such as dimethoate, the MIP-SERS substrate undergoes a binding event to generate Raman vibrational fingerprints with signal amplification of both the SERS active element (silver nanoparticles) and the dimethoate analyte

[0012] , Thus, to gain an understanding of how the synergistic effect of MIP-SERS enhances the detection of the target analyte, individual SERS measurements were conducted on the pure dimethoate and synthesized AgNPs independently as elucidated in FIG. 25 (A) and FIG. 25 (B) respectively. The SERS spectrum provided information about the characteristic fingerprints of the dimethoate and the AgNPs and provided an inference of the possible characteristic bands to expect when the MIP-SERS substrate is employed for the detection of dimethoate in complex matrices.

[0190] 2.3.7 SERS Assay and the re -binding of AgNPs@ MIP with dimethoate concentrations

[0191] The SERS activity of both the dimethoate AgNPs @ MIP and the AgNIP were evaluated with a known amount of dimethoate of concentration (10 pg / ml) to confirm its recognition efficiency for the target analyte dimethoate (FIG. 26 (A)). It was observed that the AgNIP possessed a limited and nonsignificant SERS activity as compared to the MIP due to the absence of the template molecule Docket No.: UNCG-532-PCT

[0192] (dimethoate), with the MIP depicting a strong binding potential for the target analyte with a characteristic peak intensity at 1587 cm1(FIG. 26 (A)). In addition, a binding assessment was conducted by treating the dimethoate AgNPs @ MIP with varied concentrations of dimethoate solutions ranging from 0.005 pg / ml to 20 pg / ml (FIG. 26 (B)) confirming the sensitivity of the AgNPs@MIP substrate. A characteristic SERS band at 1587 cm1was evident in all the SERS measurements with an intense enhancement when the highest concentration of dimethoate (20 pg / ml) was bound to the AgNPs@MIP. Thus, it was noteworthy that the SERS enhancement of the dimethoate measurements was directly proportional to the dimethoate concentration. Furthermore, the incubation and contact time of the MIP with the dimethoate aided the efficient adsorption potential of the AgNPs @ MIP for its target analyte. Also, the synergistic effect of the embedded AgNPs in the matrix of the MIP was vital for the electromagnetic field enhancement of the MIIP substrate and thus generated a strong SERS signal for the sensitive detection of dimethoate pesticide [27,51J.

[0193] Also, the Raman bands of the AgNPs@MIP were consistent and significantly pronounced within the entire MIP platform with the doped silver nanoparticles enhancing the SERS signal amplification. In addition, the reported characteristic bands confirm the strong interaction between the silver nanoparticles and the dimethoate imprinted polymer. It was also evident that a characteristic fingerprint and peak region between 1500 cm1and 1600 cm1had intense SERS amplification and was related to the electromagnetic enhancement of the Ag substrate [ 14,931 within the MIP. This characteristic fingerprint in the MIP was also corroborated to be silver nanoparticles as confirmed by the Raman scattering spectra of the synthesized silver nanoparticles (FIG. 25 (B)). This fingerprint region thus, became a signature peak for the AgNPs@MIP’s rebinding event with the dimethoate template and specifically showed a significantly enhanced Raman peak at 1587 cm which was evident in all samples reacted with the dimethoate solution. It can thus, be confirmed that the MIP substrate is a good host for the dimethoate analyte and has a strong affinity and enhanced sensitivity to bind dimethoate at even trace concentrations as demonstrated by the SERS results of the binding assessments with a distinct SERS characteristic peak exhibited at 1587 cm'1by the dimethoate AgNPs @ MIP.

[0194] In addition, a quantitative relationship was also confirmed between the characteristic SERS signal intensity peak at 1587 cm-1and the evaluated dimethoate concentrations as indicated in FIG. 26 (C). Thus, a linear relationship was observed in the range for the dimethoate concentrations within the range of 5 to 0.005 pg / ml and the coefficient of determination (R2) was 0.99. From the observed SERS assessment, the limit of detection (LOD) for dimethoate by using this AgNPs @ MIP SERS technique could be down to 0.005 pg / ml. Consequently, the excellent SERS spectra demonstrated by Docket No.: UNCG-532-PCT the MIP substrate as confirmed in FIG. 26, confers it as a promising SERS substrate that has the potential of detecting a wide range of concentrations of specific target analytes in complex matrices.

[0195] 2.3.8 Interference Assessment or Selectivity of the dimethoate AgNPs@MIP with other pesticides

[0196] An interference assessment of the developed sensor was done to ascertain the selectivity of the dimethoate AgNPs@MIP in the presence of other potential OPP analytes. Thus, two other pesticides namely, chlorpyrifos and fenthion were evaluated separately and as a blend with dimethoate solution at a 5 pg / ml concentration respectively as depicted in FIG. 27.

[0197] It was observed that the dimethoate AgNPs@MIP was highly selective for dimethoate in both instances when a cocktail of chlorpyrifos and fenthion were blended with dimethoate independently and evaluated. The characteristic SERS band at 1587 cm1was evident in all cases in the presence of the interferent analyte as well. Similarly, the shape of the characierislic SERS bands was similar to those previously observed in other SERS measurements. Interestingly, it was noted that, when only chlorpyrifos and fenthion were assessed individually, no notable SERS bands or characteristic peaks were observed. However, the interference from the fenthion in the presence of dimethoate resulted in a distinct signature peak shape which still confirmed the enhanced selectivity of the developed dimethoate AgNPs@MIP for dimethoate analyte. The result confirms that the MIP sensor can distinguish between other OPP analytes within a complex matrix.

[0198] 2.3.9 Real sample application and the detection of dimethoate in food samples

[0199] The enhanced performance of the fabricated sensor was also evaluated in a complex environmental matrix such as a food sample. Thus, two food samples specifically apple juice and orange juice were both spiked with 0.5 pg / ml and 10 pg / ml of dimethoate concentration, and their SERS measurements were assessed as depicted in FIG. 28 (A) and FIG. 28 (B) respectively. The successful detection of dimethoate in the food samples was confirmed with the characteristic SERS Raman band at 1587 cm1. As previously confirmed, it was evident that the concentration of dimethoate was directly proportional to the enhancement of the characteristic SERS peak. Hence, the lowest concentration of 0.5 pg / ml yielded a lesser characteristic peak while the higher concentration of 10 pg / ml resulted in an elevated characteristic Raman peak. Generally, it is known that complex matrices such as food samples, limit the sensitivity of SERS detection, due to interferences

[0094] from the structure of the sample, however, a good MIP-SERS substrate such as the developed dimethoate AgNPs@MIP system, has a robust platform with enhanced sensitivity for the target analyte dimethoate in the apple and orange juice samples [94,95], Docket No.: UNCG-532-PCT

[0200] Table 1 Detection and removal of dimethoate from environmental samples

[0201] Sample Spiked Recovered Recovery (%) Standard Relative Standard

[0202] (pg / ml) (pg / ml) Deviation Deviation (%)

[0203] Apple Juice 0.5 0.47 94.00 0.02 4.37

[0204] Apple Juice 10 9.88 98.80 0.09 0.85

[0205] Orange Juice 0.5 0.49 98.00 0.01 1.43

[0206] Orange Juice 10 10.15 101.50 0.11 1.05

[0207] 2.4 Conclusion

[0208] A one -pot fabricated and efficient dimethoate AgNPs@MIP-SERS sensor was developed by the in-situ synthesis of AgNPs in the imprinted polymer matrix for the sensitive detection of dimethoate, an environmental pollutant. The developed MIP-SERS sensor had excellent results with a limit of detection of 0.005 pg / ml and depicted a characteristic SERS peak intensity at 1587 cm1. Also, a good linear relationship was confirmed quantitatively with concentrations of dimethoate detected by the AgNPs@MIP in the range of 20 pg / ml to 0.005 pg / ml. In addition, the AgNPsOMIP sensor possessed great SERS attributes and was highly selective against other evaluated pesticides. The sensor’s performance when tested with real samples such as spiked food samples at concentrations of 10 pg / ml and 0.5 pg / ml respectively yielded remarkable results, as well as had great recovery rates. This one-pot synthesis of this novel AgNPs @MIP sensor has promising potential for the detection of environmental contaminants in complex matrices and could be employed for practical applications.

[0209] REFERENCES:

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[0272] 91. Wei, C.; Li, M.; Zhao, X. Surface-Enhanced Raman Scattering (SERS) With Silver Nano Substrates Synthesized by Microwave for Rapid Detection of Foodborne Pathogens. Front Microbiol 2018, 9, doi: 10.3389 / fmicb.2018.02857.

[0273] 92. Abu Bakar, N.; Fronzi, M.; Shapter, LG. Surface-Enhanced Raman Spectroscopy Using a Silver Nanostar Substrate for Neonicotinoid Peslicides Detection. Sensors 2024, 24, doi:10.3390 / s24020373.

[0274] 93. Lu, J.; Cai, Z.; Zou, Y; Wu, D.; Wang, A.; Chang, J.; Wang, F.; Tian, Z.; Liu, G. Silver Nanoparticle-Based Surface -Enhanced Raman Spectroscopy for the Rapid and Selective Detection of Trace Tropane Alkaloids in Food. ACS Appl Nano Mater 2019, 2, 6592-6601, doi : 10.1021 / acsanm ,9b01493.

[0275] 94. Neng, J.; Wang, J.; Wang, Y; Zhang, Y; Chen, P. Trace Analysis of Food by Surface -Enhanced Raman Spectroscopy Combined with Molecular Imprinting Technology: Principle, Application, Challenges, and Prospects. Food Chem 2023, 429, 136883, doi :https : / / doi . org / 10.1016 / j . foodchem .2023. 136883.

[0276] 95. Pang, S.; Yang, T.; He, L. Review of Surface Enhanced Raman Spectroscopic (SERS) Detection of Synthetic Chemical Pesticides. TrAC Trends in Analytical Chemistry 2016, 85, 73-82, doi:https: / / doi.org / 10.1016 / j.trac.2016.06.017. Docket No.: UNCG-532-PCT

[0277] All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

Docket No.: UNCG-532-PCTCLAIMS1. A method for selectively detecting a target analyte in a sample, the method comprising: providing a composition comprising a molecularly imprinted polymers (MIP) matrix, wherein the MIP matrix comprises a) noble metal nanoparticles or hybrid plasmonic nanoparticles and b) activated recognition cavities comprising binding sites and cavities complementary to a target analyte; contacting the composition with the target analyte; and detecting binding of the MIP matrix to the target analyte in a sample, thereby detecting the target analyte in the sample.

2. The method of claim 1, wherein the MIP matrix comprises uniformly dispersed noble metal nanoparticles or uniformly dispersed hybrid plasmonic nanoparticles.

3. The method of claim 1, wherein the composition is a surface-enhanced Raman scattering (SERS) substrate and wherein detecting binding of the MIP matrix to the target analyte in a sample comprises detecting Raman signals.

4. The method of claim 3, wherein the Raman signals are surface-enhanced Raman spectroscopy (SERS) signals.

5. The method of claim 4, wherein the SERS signals comprise a fingerprint of the binding of the MIP matrix to the target analyte.

6. The method of claim 5, wherein the fingerprint comprises an amplified SERS signal of the MIP matrix and / or an amplified SERS signal of the target analyte.

7. The method of claim 6, wherein the intensity of amplified SERS signal of the MIP matrix and / or the intensity of the amplified SERS signal of the target analyte is directly proportional to the concentration of the target analyte in the sample.

8. The method of claim 1, wherein the noble metal nanoparticlcs arc silver nanoparticlcs or gold nanoparticles.Docket No.: UNCG-532-PCT9. The method of claim 1, wherein the hybrid plasmonic nanoparticles comprise noble metal nanoparticles and carbon nanoparticles.

10. The method of claim 1, wherein the target analyte is an environmental pollutant.

11. The method of claim 1 , wherein the target analyte is an organophosphate pesticide, herbicide, a PFAS molecule, or a heavy metal ion.

12. The method of claim 1 wherein the target analyte is malathion, chlorpyrifos, dimethoate, or parathion.

13. The method of claim 1 , wherein the target analyte is present in the sample at a low concentration ranging from 0.005 pg / ml to 50 pg / ml.

14. The method of claim 1, wherein the minimum detection limit is at least 0.005 pg / ml.

15. The method of claim 1, the method further comprising recovering the target analyte with a recovery rate greater than 90%, 91%, 92%, or 93%.

16. The method of claim 1, wherein the sample comprises complex environmental matrices.

17. The method of claim 1, wherein the sample is tap water, drinking water, river water, urine, agroecosystem sediments, a fruit, or a vegetable.

18. A composition comprising a molecularly imprinted polymers (MIPs) matrix, wherein the MIP matrix comprises: a) noble metal nanoparticles or hybrid plasmonic nanoparticles, and b) an activated recognition cavity comprising binding sites and a structural shape complementary to a target analyte.

19. The composition of claim 18, wherein the MIP matrix comprises uniformly dispersed noble metal nanoparticles or uniformly dispersed hybrid plasmonic nanoparticlcs.Docket No.: UNCG-532-PCT20. The composition of claim 18, wherein the noble metal nanoparticles or the hybrid plasmonic nanoparticles are encapsulated on the surface of the MIPs matrix and embedded within the MIPs matrix.

21. The composition of claim 18, wherein the noble metal nanoparticles are silver nanoparticles or gold nanoparticles.

22. The composition of claim 18, wherein the hybrid plasmonic nanoparticles comprise noble metal nanoparticles and carbon nanoparticles.

23. The composition of claim 18, wherein the noble metal nanoparticles are silver nanoparticles, and the M1P matrix comprises activated recognition cavities complementary to malathion.

24. A method for in-situ manufacturing a molecularly imprinted polymers (MIPs) matrix comprising noble metal nanoparticles, the method comprising: providing a homogenous mixture of a noble metal precursor molecule, a template target analyte, a functional monomer, a crosslinker, and an initiator; polymerizing / solidifying the homogenous mixture to produce a molecularly imprinted polymers (MIP) matrix comprising the noble metal precursor molecule and the template target analyte; reducing the noble metal precursor molecule into a noble metal nanoparticle in-situ within the MIP matrix, wherein the MIP matrix comprises noble metal nanoparticles or hybrid plasmonic nanoparticles; removing the template target analyte from the MIP matrix, wherein the MIP matrix comprises activated recognition cavities specific to the target analyte.

25. The composition of claim 24, wherein the functional monomer is methyl methacrylate (MMA), acrylamide, acrylic acid, glycidyl methacrylate (GMA), fumaric acid, itaconic acid or vinyl siloxanes.Docket No.: UNCG-532-PCT26. The composition of claim 24, wherein the cross-linker is ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TRIM), divinylbenzene (DVB), polyethylene glycol dimethacrylate (PEGDMA), tetraethylene glycol dimethacrylate (TEGDMA), or ethylene glycol diacrylate (EGDA).

27. The composition of claim 24, wherein the initiator is 2,2'-azobisisobutyronitrile (AIBN), benzoyl pereoxide (BPO), azobisdimethylvaleronitrile (ABDV), or 2,2’ - Azobisisovaleronitrile (AIVN).

28. The method of claim 24, wherein the MIP matrix comprises uniformly dispersed noble metal nanoparticles or uniformly dispersed hybrid plasmonic nanoparticles.

29. The method of claim 24, wherein removing the template target analyte from the MIP matrix comprises reacting the MIP matrix with an organic solvent.

30. The method of claim 24, wherein the homogenous mixture further comprises a reducing agent.

31. The method of claim 30, wherein the reducing agent is NaBIU32. The method of claim 24, wherein the functional monomer is methyl methacrylate (MMA), the initiator is 2,2'-azobisisobutyronitrile (AIBN), the cross-linker is ethylene glycol dimethacrylate (EGDMA), the noble metal precursor molecule is silver nitrate, and / or the noble metal nanoparticle is a silver nanoparticle is silver nitrate.

33. The method of claim 24, wherein the template target analyte is an organophosphate pesticide, herbicide, a heavy metal ion, or a PFAS molecule.

34. The method of claim 24, wherein the template target analyte is an environmental pollutant.

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